The overall goal of this project is to develop broad-spectrum antibiotics against Yersinia pestis and other important human pathogens. The potential use of engineered, multi-drug-resistant strains as agents of bioterrorism necessitates the development of new anti-Y. pestis therapies. This is a challenging goal, since the last novel class of broad-spectrum antibiotics was introduced over 20 years ago. NovoBiotic has a proprietary technology capable of meeting this challenge. We will obtain new antimicrobials from a previously inaccessible source - unculturable microorganisms that make up 99% of the total microbial diversity. In Phase I, our goal was to develop an industrial-scale technology to grow and screen previously unculturable microorganisms for antimicrobial production. The main milestone was to "domesticate" unculturable organisms to enable scale-up under conventional growth conditions; and developing a screen capable of detecting antimicrobial activity at a rate of equal to or >10(exp5) isolates/year. We achieved these milestones ahead of schedule, and have in place a robust technology that will enable us to discover new antibiotics in this Phase II project. Our preliminary results indicate that unculturable microorganisms produce novel, broad- spectrum antimicrobial compounds. The anti-infectives we develop will target a global $24 billion market. Broad-spectrum antibiotics are the top earners, with several drugs having sales in excess of $1 billion. Phase II Specific Aims are: 1. Large-scale isolation of lead antimicrobial compounds from unculturable bacteria. Milestone: obtain 200 purified, potentially novel lead compounds. 2. In vitro validation and structure of lead compounds. a. Structure determination. All compounds derived from the bioassay guided fractionation will have their structures elucidated. We will define the basic molecular architecture using advanced MS and NMR. b. Biological validation. We will determine the following properties of the purified early leads: potency (MIC) against a panel of test organisms; MBC; activity in the presence of serum; toxicity against animal cells; resistance development; specificity of action. Milestone: obtain 50 leads with novel structure. 3. In vivo validation. The lead compounds that pass preliminary validation will be subjected to a detailed evaluation in an animal model. Milestone: obtain 3-5 advanced validated leads for drug development. Relevance to human health: There is an acute need for new antibiotics to combat conventional and biowarfare drug-resistant pathogens. The compounds we discover in this project will lead to new, broad- spectrum antibiotics. [unreadable] [unreadable] [unreadable]